Live cell imaging of β-tubulin mRNA reveals spatiotemporal expression dynamics in the filamentous fungus Aspergillus oryzae

In filamentous fungi, microtubules are important for polar growth and morphological maintenance and serve as rails for intracellular trafficking. The molecular mechanisms associated with microtubules have been analyzed. However, little is known about when and where tubulin, a component of microtubules, is biosynthesized in multinuclear and multicellular filamentous fungi. In this study, we visualized microtubules based on the enhanced green fluorescence protein (EGFP)-labeled α-tubulin and β-tubulin mRNA tagged by the EGFP-mediated MS2 system in living yellow Koji mold Aspergillus oryzae cells in order to understand the spatiotemporal production mechanism of tubulin. We found that mRNA of btuA, encoding for β-tubulin, localized at dot-like structures through the apical, middle and basal regions of the hyphal cells. In addition, some btuA mRNA dots showed microtubule-dependent motor protein-like dynamics in the cells. Furthermore, it was found that btuA mRNA dots were decreased in the cytoplasm just before mitosis but increased immediately after mitosis, followed by a gradual decrease. In summary, the localization and abundance of β-tubulin mRNA is spatiotemporally regulated in living A. oryzae hyphal cells.

specific sites in the cell is important to ensure that individual proteins are in the right place and is a necessary physiological function to prevent mislocalization of proteins 22 .In addition, mRNAs localized to specific sites can be translated multiple times, saving the energy needed to transport protein 23 .Furthermore, mRNA localization control is thought to be important for local protein synthesis 24 , and especially in multicellular organisms, disruption of proper mRNA localization has been reported to cause significant developmental and growth abnormalities [25][26][27] .The molecular mechanisms of mRNA localization are being analyzed with a focus on its physiological significance, but many aspects remain unknown.
The yellow Koji mold Aspergillus oryzae is a filamentous fungus widely used in the fermentation and brewing industries and is capable of secreting and producing large amounts of various useful enzymes safely, so studies on membrane traffic including the secretory pathway are well established 28,29 .Such studies have shown that microtubules are important in A. oryzae as rails for intracellular material transport 30 .Hydrolytic enzymes, such as amylases, which are abundantly produced in A. oryzae, are transported to secretory sites such as hyphal tips and septa in the presence of microtubules, and proper formation of microtubules is important for A. oryzae to efficiently produce secretory proteins out of the cell 31 .In A. oryzae, the localization of each mRNA and translated protein, including α-amylase and actin, was analyzed by single-molecule fluorescence in situ hybridization 32,33 .Furthermore, mRNA for glucoamylase (GlaA), one of the major secretory proteins in A. oryzae, was analyzed by the MS2 system, which enables mRNA to be visualized in living cells, revealing that glaA mRNA is transcribed from the nucleus near the hyphal tip and septum, the sites of secretion 34 .However, filamentous fungi, including A. oryzae, are multinuclear and multicellular, and the mechanisms that regulate the spatiotemporal expression of proteins that function intracellularly, such as tubulin, are largely unknown.Therefore, in this study, we analyzed the spatiotemporal dynamics of tubulin mRNA, which is a component of microtubules.

Microtubule dynamics in A. oryzae hyphal cells
In filamentous fungal cells, microtubules have been shown to exhibit dynamics in the cytoplasm, but in A. oryzae they have not been analyzed in detail.Thus, we fused enhanced green fluorescence protein (EGFP) to the N-terminus of AtuA (AO090005000840), an α-tubulin in A. oryzae, and also labeled the nucleus with red fluorescence protein mCherry-tagged nuclear localization signal (NLS).EGFP-AtuA was visualized as filaments in the cytoplasm from the basal to the tip regions (Fig. 1A).Its localization pattern is similar to its homolog protein previously seen in A. nidulans, suggesting similar microtubule formation mechanisms and functions 35 .
To further investigate the dynamics of elongated microtubules in the cytoplasm, time-lapse analysis at 2-s intervals revealed large fluctuations of microtubules throughout the cytoplasm and elongation of filament structures regardless of the hyphal regions (Fig. 1B, C, Supplementary videos S1-3).Focusing on the middle part of the hyphal cells, studies in A. nidulans have reported the presence of sMTOCs as microtubule-forming centers localized at the septum and microtubules extending from sMTOCs to the cytoplasm 13 .Furthermore, in Neurospora crassa, microtubules have been observed penetrating the septal pore at the center of the septum 36 .In A. oryzae, multiple microtubule filaments were present near the septum, and they elongated starting from the septum, suggesting the presence of sMTOC.EGFP-AtuA also diffused into the cytoplasm upon nocodazole treatment, an inhibitor of microtubule polymerization, indicating that EGFP-AtuA indeed constitutes microtubules (Fig. 1D).

Visualization of btuA mRNA by the MS2 system in living A. oryzae cells
There have been no reports of live cell imaging of mRNA encoding tubulin, a component of microtubules, in any cell type.Therefore, in order to analyze tubulin mRNA and microtubules simultaneously in A. oryzae, we decided to analyze microtubules with α-tubulin AtuA as described above and visualize β-tubulin mRNA.The MS2 system is often used to visualize mRNA in living cells, and in fact, mRNA for glaA, which encodes glucoamylase, a secreted enzyme, has been visualized and analyzed in A. oryzae 17,34 .We introduced 24 copies of the aptamer MS2-binding sites (MBS) downstream of the endogenous btuA (AO090009000281) encoding β-tubulin and generated a btuA-MS2 strain that expresses two copies of EGFP fused to MS2-coat protein (MCP), which binds specifically to MBS (Fig. 2A).To eliminate background signals when analyzing cytoplasmic btuA mRNA, NLS was added to the N-terminus of MCP-2×EGFP so that NLS-MCP-2×EGFP localizes to the nucleus when btuA mRNA is not expressed (Fig. 2A).When mCherry-NLS was also expressed to label the nucleus, btuA mRNA showed dot-like fluorescence in the cytoplasm in the btuA-MS2 strain, whereas NLS-MCP-2×EGFP was only localized to the nucleus in the strain without MBS (Fig. 2B).The microscopic analysis system used in this study did not detect the NLS-MCP-2×EGFP signal in the cytoplasm.In addition, although Z-stack was not used, the THUNDER Imager Live Cell system used in this study succeeded in obtaining clear images by computational clearing.Cytoplasmic dot fluorescence was not observed after treatment with the transcriptional inhibitor actinomycin D (Fig. 2C, D).These results suggest that the cytoplasmic dot fluorescence observed in the btuA-MS2 strain is derived from btuA mRNA.
Analysis of btuA expression by quantitative reverse transcription PCR (qRT-PCR) showed that the amount of btuA mRNA in the btuA-MS2 strain was approximately 65% lower than in the control strain (Fig. 2E).In general, it has been reported that the addition of MBS sequences in the MS2 system reduces mRNA stability 37 , suggesting that about two-thirds of the btuA mRNA was degraded in the btuA-MS2 strain.However, comparative growth analysis of the btuA-MS2 strain revealed that the reduction in btuA mRNA expression did not have a marked effect on growth (Fig. 2F, G), so we decided to use the btuA-MS2 strain for future analysis.

btuA mRNA exists through the A. oryzae hyphal cells
To determine how btuA mRNA is distributed in A. oryzae hyphal cells, we performed a localization analysis of btuA mRNA in each hyphal region.We analyzed the btuA-MS2 strain by introducing the mCherry-NLS construct into the btuA-MS2 strain to label the nuclei (Fig. 3A).First, we analyzed whether there was a gap in the number of nuclei in each hyphal region.The results showed that there was no significant difference in the number of  www.nature.com/scientificreports/nuclei in each hyphal region, and the number of nuclei was almost the same (Fig. 3B).Then, we quantified the number of dots shown by btuA mRNA and found that btuA mRNA was almost uniformly localized in the cytoplasm, regardless of the hyphal regions (Fig. 3C).The number of btuA mRNA dots per number of nuclei also did not differ among hyphal regions (Fig. 3D).These results indicate that btuA mRNA is uniformly expressed and present regardless of the hyphal regions.

The dynamics of btuA mRNA are dependent on microtubules
It has been shown that in A. oryzae, a portion of the mRNA for glaA, which encodes the secretory protein glucoamylase, migrates long distances on microtubules in a kinesin motor-dependent manner 34 .Therefore, nocodazole treatment, a microtubule polymerization inhibitor, was first applied to analyze the microtubule dependence of btuA mRNA dynamics.The results showed that in the control DMSO treatment, btuA mRNA exhibited motor protein-like kinetics with linear long-distance movement after migration from the nucleus to the cytoplasm (Fig. 4A, B, Supplementary videos S4, S5).Assuming that such long-distance dynamics are microtubule-dependent, we quantified the linear migration distance of btuA mRNA monitored by tracking analysis and showed that nocodazole treatment shortened the displacement of btuA mRNA (Fig. 4C).Reviewing the data on 300 btuA mRNA movements, as shown in Fig. 4C, long-range movement as in Fig. 4B was less common, and long-range and bi-directional movement was not observed.These results indicate that btuA mRNA, like glaA mRNA, exhibits microtubule-dependent long-range dynamics.

The abundance of btuA mRNA in the cytoplasm is dependent on cell cycle
Microtubules dynamically change their localization in a cell cycle-dependent manner.Time-lapse analysis of A. oryzae strains labeled with microtubules and nuclei in mitosis clearly showed that microtubules formed spindles at the timing of nuclear division, followed by the re-formation of doubled nuclei and cytoplasmic microtubules within 10 min (Fig. 5A).Then, we analyzed btuA mRNA in mitosis and found that the number of btuA mRNA dots in the cytoplasm decreased just before mitosis, reached a maximum at the time of mitosis, and then slowly decreased (Fig. 5B, C).To confirm that the decrease in btuA mRNA in the cytoplasm after mitosis was not due to fading of the fluorescent protein, because of the long time-lapse analysis, we observed a 30-min interval immediately after mitosis and obtained similar results (Fig. 5D).To confirm whether the observed cytoplasmic www.nature.com/scientificreports/dot fluorescence was actually indicative of btuA mRNA, we observed NLS-MCP-2xEGFP in the strain lacking MBS.During mitosis, NLS-MCP-2xEGFP diffused into the cytoplasm and showed no dot-like localization, but upon return to interphase it again showed nuclear localization, indicating that the dot fluorescence observed in the btuA-MS2 strain was derived from btuA mRNA (Fig. 5E).These results demonstrate that btuA mRNA changes its amount in the cytoplasm in response to the cell cycle.

Discussion
To our best knowledge, tubulin mRNA has never been reported to be visualized in living cells of any species.Therefore, in this study, we attempted to visualize A. oryzae live cells and found that the MS2 system enables visualization of btuA mRNA, which encodes β-tubulin, and that spatiotemporal expression dynamics exist.btuA mRNA was shown to be uniformly expressed from the basal to the tip regions and localized throughout the cytoplasm of hyphal cells.It is conceivable that the btuA mRNA found in the nucleus could be present in the nucleus or also localized to the SPB.It has been analyzed in A. nidulans with ApsB as a marker for SPB 38 , and future co-localization analysis with the A. oryzae ortholog AoApsB (AO090020000040) is possible.
The quantitative data on the number of nuclei and cytoplasmic mRNA suggested that the same amount of btuA mRNA was expressed per nucleus at all sites in the hyphal cells.This mode of expression of btuA mRNA differed from the finding that glaA mRNA, encoding the secretory protein glucoamylase, is transcribed predominantly in nuclei near the hyphal tip and septum 34 .In filamentous fungi, secretory proteins are secreted from the hyphal tips and septa 31 , so it makes sense that glaA mRNA is preferentially expressed in nuclei near such sites.On the other hand, if we consider that microtubules exhibit dynamics throughout the mycelial cell and are repeatedly turned over at the protein level, this is consistent with the fact that btuA-encoded β-tubulin mRNA is expressed in hyphal regions without being restricted to them.Microtubules serve as rails for hyphal cell morphology maintenance and secretory vesicle and early endosomal transport 30 .This is consistent with the need for a constant and extensive supply of tubulin mRNA for microtubule function not limited to specific hyphal regions.These results suggest that proteins secreted extracellularly and those functioning intracellularly are spatially regulated by different mechanisms.
The kinetic analysis of btuA mRNA suggested the involvement of microtubules in its localization.Indeed, in vitro analysis of mouse β-tubulin mRNA reported that β-tubulin mRNA is regulated by RNA-binding protein (RBP)-mediated transport by kinesin motor proteins 39 .Thus, it is expected that in A. oryzae, btuA mRNA also undergoes kinesin motor-dependent transport regulation.To further investigate the association between β-tubulin mRNA localization and microtubules in A. oryzae, we need to attempt to identify RBPs that interact www.nature.com/scientificreports/with β-tubulin mRNA and RBP binding sites on mRNA.Many proteins are involved in microtubule regulation as microtubule-interacting proteins, and it is necessary to analyze the extent to which β-tubulin mRNA is also regulated by RBPs in the future.
In mitosis, microtubules dynamically change their localization.Time-lapse analysis focusing on the cell cycle revealed that in A. oryzae, spindle formation occurs simultaneously in multiple nuclei, and that the disappearance and formation of cytoplasmic microtubules are carried out in a short period of about 10 min before and after the spindle formation.This phenomenon has also been observed in A. nidulans and is reported to proceed at a rate comparable to the mitosis observed here in A. oryzae 40 .We found that btuA mRNA in the cytoplasm increased rapidly during mitosis, followed by a gradual decrease.It is likely that translation occurs in response to the increasing amount of btuA mRNA, resulting in the de novo synthesis of β-tubulin.However, the molecular regulation mechanism and physiological significance of this phenomenon are currently unknown.The SunTag method is a technique to visualize translated mRNA [41][42][43][44][45] , and the degree of btuA mRNA translation should also be analyzed in the future.In addition, further work should attempt to clarify how tubulin mRNA localization throughout the cytoplasm and cell cycle-dependent expression changes revealed by this study are related to tubulin production and microtubule formation.

Plasmid and strain construction
The A. oryzae strains used in this study are listed in Table 1.Genomic DNA of the wild-type A. oryzae strain RIB40 was used as the template for the DNA cloning.For visualization of microtubules, the plasmid pgPaEGAtuA 34 for expression of EGFP-AtuA was transformed into the NSlD1 strain to create the GaA1 strain.To further visualize nuclei, the plasmid pgPtmCN 34 expressing mCherry-NLS was introduced into the GaA1 strain to create the GaA_CN1 strain.

Imaging analysis
Counting of btuA mRNA dots was performed using the RS-FISH plugin in ImageJ Fiji 48,49 as described previously 34 .The images of btuA mRNA and nuclear localization were converted from 16 to 8 bit and merged, and each hyphal region was cropped.Then, each image was re-segmented and images of nuclei were binarized and subtracted from those of btuA mRNA using the image calculator.The background fluorescence of the btuA mRNA images, in which nuclear fluorescence was eliminated, was completely subtracted by the Math function and processed with a Gaussian filter.Measurements with RS-FISH were performed with the following settings: In Radial Symmetry, Mode = Interactive, ZYX = 1.000;RANSAC was set; Use anisotropy coefficient for Dog was checked; and Spot intensity was set to Linear Interpolation.Then, btuA mRNA counts were measured with Adjust difference-of-gaussian values set to sigma = 1.5 and Threshold = 0.0066.For tracking analysis, each time-lapse image was acquired for 20 s in the region of 50 µm from the hyphal tip and processed using the THUNDER imaging system described above.Then, ImageJ Fiji was used and btuA mRNA dynamics was analyzed using the TrackMate plug-in.The Dog Detector was used and the threshold was set to 0.3 µm to detect only intracellular-specific fluorescence.Thereafter, Lap Tracker was run with the distance between frames set to 0.8 µm, the gap distance between the three frames set to 0.8 µm and the Track displacement threshold set to 0.5 µm or greater.

Figure 1 .
Figure 1.Dynamics of microtubules in A. oryzae hyphal cells.(A) Colocalization of microtubules (EGFP-AtuA, green) and nuclei (mCherry-NLS, magenta).White arrowheads point to septa.(B) Time-lapse images of (A) were taken at 2 s intervals, and images at 0 s, 4 s, 8 s and 12 s are shown.White dot lines indicate septa.(C) Enlarged time-lapse images of the yellow frames in (B).Yellow arrowheads indicate elongating microtubule ends at each region.(D) Localization of microtubules in cells treated with DMSO or nocodazole.Scale bars, 10 µm.

Figure 2 .
Figure 2. Visualization of butA mRNA by the MS2 system in A. oryzae living cells.(A) Scheme of strategy for visualization of btuA mRNA by the MS2 system.24×MBS was inserted into the 3′UTR of endogenous btuA gene and NLS-fused MCP-2×EGFP was co-expressed.(B) Visualization of MCP-2×EGFP or btuA mRNA (green) and nuclei (magenta) in the apical cells.In the enlarged image on the right, white arrowheads point to btuA mRNAs localized in the cytoplasm.Scale bars, 10 µm.(C) Visualization of btuA mRNAs (green) and nuclei (magenta) in cells treated with DMSO or actinomycin D. Scale bars, 10 µm.(D) The number of btuA mRNA dots localized in the cytoplasm in each culture condition.Error bars indicate standard deviation of the mean (n = 5).Student's t-test, no significant difference at 0 h (p = 0.1075).(E) Relative expression level of btuA mRNA.Error bars indicate standard deviation of the mean (n = 3).Student's t-test, no significant difference between the control and MCP strains (p = 0.2604).(F) Growth tests of the control, MCP and btuA-MS2 strains.(G) Diameter of colony in each strain.Error bars indicate standard deviation of the mean (n = 3).

Figure 3 .
Figure 3. Distribution of btuA mRNA in A. oryzae hyphal cells.(A) Localization of btuA mRNA (green) and nuclei (magenta) in each hyphal area.Scale bars, 10 µm.(B-D) The number of nuclei (B), btuA mRNA dots (C) and btuA mRNA dots/nuclei (D) in each hyphal area.Tukey-Kramer test does not show significant difference among hyphal regions.Error bars indicate standard deviation of the mean (n = 5).

Table 1 .
Strains used in this study.